James W. Campbell

Phosphoenolpyruvate carboxylase activity and glycogenesis in the flatworm, hymenolepis diminuta

Abstract 1. 1. Hymenolepis incorporates C 14 into organic, keto and amino acids and into polysaccharide when incubated with NaHC 14 O 3 and glucose. There is a marked decrease in CO 2 incorporation into polysaccharide under anaerobic conditions. This indicates the presence of an energy-dependent link between the glycotic pathway and the C 4 -dicarboxylic acid sequence of the tricarboxylic acid cycle. 2. 2. Phosphoenolpyruvate carboxylase, which serves as this connecting reaction in avian and mammalian tissues, is the most active CO 2 -fixing enzyme in Hymenolepis . It is localized mainly in the soluble fraction although some activity is associated with the particulate fractions. There is an increase in this carboxylase activity concomitant with an increase in CO 2 incorporation into polysaccharide. This indicates that the enzyme may also function in this flatworm to convert dicarboxylic acids to phosphopyruvate during gluconeogenesis. 3. 3. Malic acid appears to be the major dicarboxylic acid formed by independent CO 2 -fixing reactions in Hymenolepis . Its formation is through the action of a “malic enzyme” and possibly also by a separate reaction. Malate synthesis is localized in the particulate fractions. Pyruvate carboxylase and propionyl-CoA carboxylase activities could not be detected.

Arginine and urea biosynthesis in the earthworm Lumbricus terrestris

Abstract 1. 1. NaHC14O3, ureido-C14-citrulline and 5-C14-ornithine were shown to be effective precursors of protein arginine in Lumbricus terrestris. C14 from NaHC14O3 and from ureido-C14-citrulline is incorporated into the guanidino-C, and from 5-C14-ornithine into the ornithine moiety of protein arginine. C14 from both NaHC14O3 and ureido-C14-citrulline is also incorporated into excretory urea. 2. 2. Lumbricus, which appears to normally be ammonotelic, becomes ureotelic when starved. There is a concomitant increased incorporation of bicarbonate-C14 into urea during this transition. In fed worms, citrulline formation is the limiting step in urea biosynthesis. During starvation, citrulline utilization becomes limiting. 3. 3. All five enzyme-catalyzed reactions of the urea cycle have now been demonstrated with cell-free preparations of Lumbricus gut tissue. The rates measured for these reactions in vitro will account for the total urea formed by whole worms. The increased formation of citrulline during starvation is due mainly to increased activity of ornithine transcarbamoylase as well as an increase in carbamoylphosphate synthetase activity. 4. 4. The earthworm thus possesses a complete urea cycle which may normally function to synthesize arginine. In the transition from ammonotelism to ureotelism during starvation, the cycle may then be utilized to detoxify the excess ammonia produced by increased protein catabolism.

A comparative study of molluscan and mammalian arginases

Abstract 1. 1. Certain properties of molluscan arginase, purified from the hepatopancreas of Otala lactea, were compared with those of rat liver arginase. 2. 2. The two enzymes showed a great deal of similarity in those properties associated with the basic catalytic mechanism such as substrate specificity, substrate binding and inhibition by basic amino acids. 3. 3. They differed mainly in their general protein properties: the snail enzyme was anionic during starch gel electrophoresis at pH 7 while the rat enzyme was cationic. 4. 4. A comparison of the divalent ion activation of the proarginases from each species was also made.

UTILIZATION OF C14O2 FOR THE FORMATION OF SOME AMINO ACIDS IN THREE INVERTEBRATES.

Abstract 1. 1. Three invertebrate organisms: the oyster ( Crassostrea virginica ), the land snail ( Otala lactea ) and a brackish water clam ( Rangia cuneata ), were investigated for their ability to incorporated carbon dioxide into the carbon chain of amino acids. 2. 2. C 14 O 2 was effectively incorporated into alanine, aspartate and glutamate by all three organisms although at different rates. 3. 3. Pyruvate-2-C 14 was also incorporated into the same three amino acids. Aspartate-U-C 14 gave rise to labeled glutamate and glutamate-U-C 14 gave rise to labeled aspartate indicating full operation of the citric acid cycle. 4. 4. Transminases linked with the citric acid cycle were measured. The activities of these enzymes can easily account for rapid deamination and reamination of the three amino acids and their corresponding keto acids respectively.

Arginine biosynthesis and metabolism in terrestrial snails

Abstract 1. 1. Arginine synthesis was studied by the incorporation in vivo of [C14]-labeled precursors into protein arginine in Otala (= Helix) lactea, Helix aspersa and Bulimulus alternatus. The pattern of incorporation and the specific conversions obtained with these precursors were consistent with the pathway for arginine biosynthesis as it is known in other organisms. 2. 2. A comparison of the incorporation of [C14]bicarbonate into protein arginine, aspartate and glutamate indicated that the rate of arginine synthesis in vivo is significant in supplying arginine for protein synthesis. 3. 3. Arginine degradation in O. lactea and H. aspersa tissues was shown to be due to the combined action of arginase, urease and δ-ornithine transaminase. Because of the action in vivo of urease, there is a rapid turnover of urea in these two species. Urease is absent from B. alternatus hepatopancreas and the urea formed from arginine in this tissue accumulates.

Studies on tissue arginase and ureogenesis in the elasmobranch, Mustelus canis.

Abstract Fourteen tissues of the elasmobranch, Mustelus canis , have been examined for arginase activity. Of these tissues, liver, kidney, and rectal gland were found to contain the highest activities, in that order. The liver arginase system was examined in more detail and was found to be similar to other vertebrate arginase. Kinetic data were obtained for the liver, kidney, and rectal gland, and the apparent K m values of these systems showed a decrease with decreasing amounts of activity. The blood levels of arginine were determined for M. canis and several other elasmobranchs and were not found to differ considerably from other vertebrates. The possible role of the tissue arginase systems in the extrahepatic synthesis of urea in elasmobranchs is discussed.

Studies on urea cycle enzymes in the terrestrial snail, Otala lactea☆

Abstract The hepatopancreas tissue of the land snail, Otala lactea, has been investigated for the presence of the enzymes known to be involved in the biosynthesis of urea in vertebrates and microorganisms. Ornithine transcarbamylase and argininosuccinate-splitting enzyme activity have been detected in this species for the first time in an invertebrate animal. Although the snail ornithine transcarbamylase was shown to require carbamyl phosphate for activity, the synthesis of this compound could not be demonstrated in vitro. Citrulline metabolism by extracts of the hepatopancreas was found to have similar requirements to the argininosuccinate synthetase system of vertebrates, and suggestive evidence for the presence of this or a very similar enzyme system in the snail was obtained. Studies on the arginase activity of the hepatopancreas showed that estivation did not significantly affect the tissue arginase levels in this species. A small amount of urease activity was also demonstrated in extracts of the hepatopancreas.

Purine biosynthesis and excretion in Otala (=helex) lactea: An evaluation of the nitrogen excretory potential

Abstract 1. 1. The accumulation of uric acid, xanthine and guanine in estivating ] Otala lactea kidneys was used to estimate the rate of biosynthesis and deposition of the kidney purines in vivi . This estimated rate was 0.18 μmole purine per g total wt. per day. 2. 2. The rate of synthesis and deposition of the kidney purines in active snails was estimated by measuring the rate of incorporation in vivo of [1-C 14 ]glycine into the kidney purines. The highest observed rate was 0.68 μmole purine per g total wt. per day. 3. These estimated rates would account for the potential of excreting 9.7 mg N (693 μatom N)/kg total wt. per day as purine-N in estivating snails and 39.1 mg N (2793 μatom N)/kg total wt. per day in active snails. The term “recretion” is used here to apply to the depostion of the purines in the kidneys and not necessarily to their elimination from the body. 4. 4. Purines are the major nitrogenous excretory product of O. lactea since purine-N makes up roughly 90 per cent of the total N present in whole kidneys of both active and estivating snails. The only other major form of N eliminated by O. lactea is NH 3 gas. This was shown previously to arise from the action in vivo of tissue urease. From the estimated rates of purine-N excretion, the NH 3 -N eliminated would account for no more than about 30 per cent of the total N excreted by estivating snails and no more than about 5 per cent of the total N excreted by active snails.

Distribution of arginase activity in mollusks

Abstract 1. 1. In the seventy species of mollusks examined, arginase activity was of most common occurrence in the pulmonate gastropods, being present in terrestrial, fresh-water and marine species. Only in this group of molllusks did levels of activity exceed those found in vertebrate liver. Arginase occurred less frequently in bivalves and was present in low levels only in some fresh-water and estuarine species. It was not detected in eleven marine species. Activity was not detected in a single amphineuran, but was present in two species of cephalopods. The occurrence of arginase in mollusks appears to be influenced more by taxonomic relationships than by either habitat or feeding habits. 2. 2. In several species possessing arginase activity, the enzyme was not restricted to the hepatopancreas tissue. Arginase activity was present in all tissues examined from a land snail and octopus. 3. 3. The levels of activity in the hepatopancreas of the land snail Otala were extremely variable among individuals of a given population kept under uniform laboratory conditions. The cause of this variation was not established. Prolonged starvation appeared to cause a slight increase in activity, while injury or estivation caused a decrease.

Ornithine transcarbamylase and arginase activity in flatworms

Abstract 1. 1. Fifteen species of flatworms, representing the three classes of the phylum and several environments, were examined for the presence of the urea cycle enzymes, ornithine transcarbamylase and arginase. 2. 2. Ornithine transcarbamylase activity was demonstrated in several species of each class and from each of the environments. In comparison with the other flatworms, an elasmobranch parasite, Lacistorhynchus tenuis , was shown to have exceptionally high levels of this activity. 3. 3. Arginase activity was found in all species of cestodes and trematodes and in the land planarian: it was not detected in fresh-water planarians and a single species of marine turbellarian.